The inhibition of restriction endonuclease PvuII cleavage activity by methylation outside its recognition sequence.

The recombinant plasmid pGEM4Z-ras DNA which was methylated on dam and dcm sites outside the PvuII recognition sequence was digested with restriction endonuclease PvuII, and one of the three PvuII sites was about 16-fold less efficient to cleave than either of the other two. On the contrary, the three PvuII sites were cleaved at about the same rate on the unmethylated DNA molecule. The results show that the cleavage inhibition of the methylated DNA on the certain PvuII site was caused by methylation outside the PvuII recognition sequence. Maybe a adjacent methylated dam site *A was responsible for the less efficient cleavage. This observation suggests that methylation outside the recognition sequence may be considered a new factor in the kinetic experiment of restriction endonuclease.     

The effect of methylation outside the recognition sequence of restriction endonuclease PvuII on its cleavage efficiency.

This study is to extend our earlier observation that Dam and Dcm methylation outside the PvuII recognition sequence inhibited PvuII cleavage in one of the three PvuII sites of pGEM4Z-ras DNA. In this paper, a new recombinant plasmid DNA, pGEM4-SV40ori-anti-ras, was constructed which has only two PvuII sites, I and II. The Dam and Dcm-methylated and unmethylated DNAs were produced in Escherichia coli and linearized by ScaI. The DNA molecules were digested with different amounts of PvuII. The results show that by comparing the DNA fragment number and intensity of the partial and final products in agarose gel, PvuII site I on the methylated DNA molecule was digested four- to eight-fold more slowly than site II. In the unmethylated plasmid DNA, the two PvuII sites were cleaved at about the same rate. The difference was caused only by methylation of Dam and Dcm sites outside the PvuII recognition sequence. A methylated Dam site immediately adjacent to the less efficiently cut PvuII site I may be responsible for the inhibitory effect. We suggest that a new parameter, involving methylation of sites outside the recognition sequence, be considered in kinetic experiments on cleavage.     

Primary sequence and partial secondary structure of the 12S kinetoplast (mitochondrial) ribosomal RNA from Leishmania tarentolae: conservation of peptidyl-transferase structural elements.

The sequence of the 1173 nt 12S kinetoplast ribosomal RNA from Leishmania tarentolae was determined from the maxicircle DNA sequence, and the 5' and 3' ends localized by primer runoff and S1 nuclease protection experiments. The gene was shown to be free of introns by S1 nuclease analysis. A partial secondary structure model of the 12S RNA molecule is presented which is equivalent in certain respects to the corresponding portions of the Escherichia coli 23S ribosomal RNA model. Domain II of the E. coli model is completely missing in the kinetoplast model with the exception of several phylogenetically conserved stems and one loop. There is a striking conservation of the functionally important peptidyl-transferase region except for the deletion of a few stems and loops. The 12S RNA is the smallest large subunit ribosomal RNA described to date.     

Human 18 S ribosomal RNA sequence inferred from DNA sequence. Variations in 18 S sequences and secondary modification patterns between vertebrates.

We have determined the DNA sequences encoding 18 S ribosomal RNA in man and in the frog, Xenopus borealis. We have also corrected the Xenopus laevis 18 S sequence: an A residue follows G-684 in the sequence. These and other available data provide a number of representative examples of variation in primary structure and secondary modification of 18 S ribosomal RNA between different groups of vertebrates. First, Xenopus laevis and Xenopus borealis 18 S ribosomal genes differ from each other by only two base substitutions, and we have found no evidence of intraspecies heterogeneity within the 18 S ribosomal DNA of Xenopus (in contrast to the Xenopus transcribed spacers). Second, the human 18 S sequence differs from that of Xenopus by approx. 6.5%. About 4% of the differences are single base changes; the remainder comprise insertions in the human sequence and other changes affecting several nucleotides. Most of these more extensive changes are clustered in a relatively short region between nucleotides 190 and 280 in the human sequence. Third, the human 18 S sequence differs from non-primate mammalian sequences by only about 1%. Fourth, nearly all of the 47 methyl groups in mammalian 18 S ribosomal RNA can be located in the sequence. The methyl group distribution corresponds closely to that in Xenopus, but there are several extra methyl groups in mammalian 18 S ribosomal RNA. Finally, minor revisions are made to the estimated numbers of pseudouridines in human and Xenopus 18 S ribosomal RNA.     

Cloning and characterization of the ribosomal genes of the sea-urchin Paracentrotus lividus. Heterogeneity of the multigene family

A Paracentrotus lividus genomic library was constructed using sperm DNA prepared from a single animal. The DNA was fragmented by partial digestion with DNase II, sized on a preparative agarose gel and inserted in the Pst I site of pBR 322 by the dG X dC tailing method. Recombinant plasmids containing ribosomal DNA were isolated, a restriction map of the gene was determined and the 18S and 26S transcribed sequences were located by S1 protection mapping. The organization of the ribosomal genes in genomic DNA of individual animals and of a pool of animals was studied by blot-hybridization of the restriction fragments, using as probes nick-translated 32P-labelled cloned ribosomal DNA fragments or 18S and 26S sea-urchin ribosomal RNA. The repeat length of the ribosomal unit was about 10.5 X 10(3) bases. A comparison of the restriction patterns of DNA from different animals showed a marked sequence heterogeneity in the spacer region of these genes. Variations of about 200 base pairs were detectable in the length of the spacer of some individuals.     

Phylogenetic relationships of three species of crows (Corvidae, Aves) based on the restriction site variation of nuclear ribosomal RNA gene

Southern blot analysis of nuclear ribosomal DNA (rDNA) was carried out to examine phylogenetic relationships between three species of crows: Corvus cornix, C. corone and C. macrorhynchos. In this purpose DNA samples of birds were digested by 12 restriction enzymes (EcoRI, HindIII, PstI, BamHI, DraI, PvuII, KpnI, XbaI, BglII, BclI, SacI and AatI) and hybridized with the clones of mouse rDNA probes (18S, 28S and INT). Based on the data obtained and Gallus gallus restriction map as a standard the restriction site maps of the main rDNA repeating unit types (repetypes) were constructed. The length of crow rDNA genes was estimated to be 22.5 kb for Jungle and 22.0 kb for Hooded and Carrion crows. C. corone and C. cornix shared a common repetype which differed, by presence of two restriction sites (XbaI and PvuII) in the spacer region, from that of C. macrorhynchos with the estimated sequence divergence of 0.26%. Restriction-size variation was revealed between individuals of C. corone and C. cornix, although the substantial meanings of this variation remain unclear yet. These data suggest that the crow species evolve with slower rate of molecular evolution, as generally observed in other avian species, compared with the higher extent in external morphology, ecological features and behavior.     

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.     

Isolation and sequence organization of human ribosomal DNA.

The genes coding for 28 S and 18 S ribosomal RNA have been purified from leukemic leukocytes of one human individual by density gradient centrifugation. The purified ribosomal DNA was analyzed by restriction endonuclease digestion and electron microscopy. The location of cleavage sites for the restriction endonuclease EcoRI was established by R-loop mapping of restriction fragments by electron microscopy. The results are in agreement with gel analysis and gel transfer hybridization. One type of ribosomal DNA repeating unit contains four cleavage sites for EcoRI. Two of these cuts are located in the genes coding for 28 S and 18 S rRNA, while the other two are in the non-transcribed spacer. Thus, one of the restriction fragments generated contains non-transcribed spacer sequences only and is not detected by gel transfer hybridization if labeled rRNA is used as the hybridization probe. A second type of repeating unit lacks one of the EcoRI cleavage sites within the non-transcribed spacer. This indicates that sequence heterogeneity exists in human rDNA spacers. R-loop mapping of high molecular weight rDNA in the electron microscope reveals that the majority of repeats are rather uniform in length. The average size of 22 repeats was 43.65(±1.27) kb. Two repeats were found with lengths of 28.6 and 53.9 kb, respectively. This, and additional evidence from gels, indicates that some length heterogeneity does exist in the non-transcribed spacer. The structure of the human rDNA repeat is summarized in Figure 10.